Device and method for detecting and localizing cells by means of photosensitive waveguides

ABSTRACT

The present invention provides devices and methods for detection of particles, such as biological cells, in samples using a photosensitive waveguide. The photosensitive waveguide changes its transmissivity in a detectable manner in response to controlling radiation emitted from the particles. In preferred embodiments, the waveguide is two-dimensional and the position of the particles as well as their presence is obtained by scanning the waveguide in two non-parallel directions. The provided devices are preferably used to locate labeled cells. The present invention also includes control systems and methods for detecting and locating cells using the devices provided.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 10/182,210 filed Jul. 22, 2002, from which the benefit ofpriority is asserted and the specification of which is incorporated byreference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of automatedbiological analysis systems and more specifically to automated celldetection systems employing waveguides having photosensitive components.

BACKGROUND

[0003] The automated detection of rare cells in a population ofdifferent cells is a challenging problem akin to finding the proverbialneedle in the haystack. Examining cell sample using traditionalmicroscopy would require unreasonable amounts of time and is susceptibleto operator error. Furthermore, in some instances, for example, thedetection of microbial cells in natural water ecosystems, thecharacteristics of the rare cells are not already known. In suchsituations optical microscopy based on image processing methods is theonly viable alternative. A similar approach may be needed when the cellsin question are known, but they are not identifiable through any othermechanism except with traditional microscopy and image processing, e.g.in the detection of cancer cells in pap-smears.

[0004] In cases where a specific characteristic of the rare cell can beidentified and located through the generation of a distinct signal,detection methods other than image processing may be employed allowingfor much faster detection in a very large initial population of cells.If, for example, a cell surface antibody specific to the cell inquestion can be bound to a fluorescing substance, the cell can bedetected using fast methods such as fluorescence activated cell sorting(FACS). In such systems though the efficiency of detection is inverselyproportional to the frequency of the cells in question.

[0005] U.S. Pat. No. 4,746,179 issued on May 24, 1988 to Dahne et al.describes the use of a waveguide in conjunction with a fluorescencesignal generated by the sample to estimate the concentration of asoluble antigen. Dahne et al. passes an excitation signal through thewaveguide that is immersed in, or in direct contact with, the samplesolution. The leakage of the excitation signal from the waveguide reactswith the solution next to the waveguide and produces a fluorescencesignal that is picked up by the waveguide and directed to and measuredby a detector. The strength of the fluorescence signal will beproportional to the concentration of the sample in solution.

[0006] Citation or identification of any reference in this section orany section of this application shall not be construed that suchreference is available as prior art to the present invention.

SUMMARY OF THE INVENTION

[0007] This present invention has for its objects devices, systems, andmethods for identification of particles of interest in lowconcentrations and with high accuracy. Not only are particles ofinterest identified, but also their spatial position in a measurementregion may be identified, permitting later verification by means ofmicroscopic image processing. Although the present invention may be usedto detect the presence and position of a wide range of particles, itsprimary application is to cell detection, wherein it improves on currentmethods of cell screening. In the following, but without limitation, theinvention will be primarily described in its principal application.

[0008] The present invention achieves its objects by means of a noveland inventive combination of a two-dimensional (2D), photosensitivewaveguide with a 2D scanning of the waveguide with the intersection ofbeams of radiation. In a photosensitive waveguide, the properties ofguided radiation change in response to incident light. Thephotosensitive waveguides of the present invention are based on noveluses of nonlinear optical (NLO) effects that take place in certainsubstances. These effects result in changes of optical properties, suchas index of refraction or absorptivity, in response to the intensity ofthe incident light. These substances can be various organic compoundse.g. conjugated dye molecules or special polymers. Such a compound maybe used as a cladding for a cylindrical or a flat optical waveguide. Abiological specimen containing various kinds of cells can be treated sothat certain cells emit radiation to which the optical properties of thecladding ate responsive. If the cells are juxtaposed to the guide, itwill affect the attributes of light radiation transferred by thewaveguide. By monitoring the changes in the properties of the lighttransferred through the waveguide, one can detect the presence of theinteresting cells.

[0009] This invention also provides for determining the location ofinteresting cells in a largely 2D measurement region by making use ofthe above detection principle. If two non-parallel beams are guidedthrough a waveguide, both will have detectable changes if an emittingcell is in proximity to the intersection of the two beams. Thus, thepresence and position of labeled cells can be determined by scanning theregion of intersection of two non-parallel beams guided through thewaveguide. In various alternatives, the waveguide may be moved or a beamof the fluorescence-inducing radiation can be scanned, or both.

[0010] In the following, a cell emitting radiation that controls aphotosensitive waveguide will cause detectable changes in the propertiesof a light beam that is guided in proximity to the emitting cell.Typically, the necessary proximity is within one to three celldiameters.

[0011] In more detail, the present invention has several embodiments. Ina first embodiment the invention includes a device for detecting thepresence of one or more particles, wherein the particles emitcontrolling radiation and are placed in a measurement region, the devicecomprising: a photosensitive waveguide, wherein one or more propertiesof radiation guided through the waveguide are responsive to controllingradiation emitted by particles present in the measurement region, and aphoto-detection system responsive to the one or more properties of theradiation guided through the photosensitive waveguide, wherein particlesemitting controlling radiation in the measurement region cause changesin the one or more properties of the radiation guided through thewaveguide which are detectable by the photo-detection system, wherebythe system detects the presence of particles.

[0012] In a second embodiment the invention includes a system fordetecting the presence and position of one or more cells which emitcontrolling radiation comprising: a measurement region in which thecells are affixed, a two-dimensional (2D) photosensitive waveguide,wherein one or more properties of radiation guided through the waveguideare responsive to controlling radiation emitted by cells present in themeasurement region, and a photo-detection system responsive to the oneor more properties of a first beam of radiation and of a second beam ofradiation, wherein the first and second beam are guided through thephotosensitive waveguide in non-parallel directions, wherein cellsemitting controlling radiation in the measurement region cause changesin the one or more properties of the first or the second beam ofradiation when the beams guided through the waveguide in proximity to anemitting cell, the changed properties being detectable by thephoto-detection system, whereby the system the presence and position ofcells.

[0013] A first aspect of the second embodiment further includes meansfor moving the first and the second beam of radiation in non-paralleldirections so that their region of intersection scans substantially allof the 2D photosensitive waveguide that is exposed to the measurementregion, and a controller for providing control signals to thephoto-detection system and to the means for moving. Further, thecontroller may include a memory, and a processor coupled to the memoryand for causing the generation of the control signals, wherein thememory contains encoded program instructions for causing the processorto perform the steps of (i) generating control signals to cause themeans for moving to move the first and the second beam of radiation sothat their region of intersection scans substantially all of themeasurement region, (ii) generating control signals to cause thephoto-detection system to detect the changed properties of the beams,(iii) storing the positions of the beams when the photo-detection systemdetects changed properties, and (iv) computing the presence and positionof cells from the stored positions.

[0014] In a third embodiment the invention includes a system fordetecting the presence and position of one or more cells, wherein thecells are labeled to emit controlling radiation in response to incidentactivation radiation, the system comprising: a two-dimensional (2D)photosensitive waveguide, wherein one or more properties of radiationguided through the waveguide are responsive to controlling radiationemitted by cells present in the measurement region, and wherein thewaveguide is planar and substantially disk shaped, and a measurementregion in which the cells are affixed, means for rotating thedisk-shaped 2D planar photosensitive waveguide together with themeasurement region, a photo-detection system responsive to the one ormore properties of a beam of radiation, wherein the beam is guidedthrough the photosensitive waveguide along a diameter of the disk-shaped2D planar photosensitive waveguide, and means for scanning a beam ofactivation radiation along the path of the beam guided though thewaveguide, wherein the activation radiation causes the labeled cells toemit controlling radiation, wherein cells emitting controlling radiationin the measurement region in response to incident activation radiationcause changes in the one or more properties of the beam of radiationwhen the beam guided through the waveguide in proximity to an emittingcell, the changed properties being detectable by the photo-detectionsystem, whereby the system the presence and position of cells.

[0015] A first aspect of the third embodiment further includes acontroller for providing control signals to the means for rotating andto the means for scanning activation radiation, where the controllerfurther includes a memory, and a processor coupled to the memory and forcausing the generation of the control signals, wherein the memorycontains encoded program instructions for causing the processor toperform the steps of (i) generating control signals to cause the meansfor rotating and the means for scanning so that the region ofintersection of the beam guided through the waveguide and the activationbeam scans substantially all of the measurement region, (ii) generatingcontrol signals to cause the photo-detection system to detect thechanged properties of the beam guided through the waveguide, (iii)storing the angular position of the waveguide and the position of thebeam of activation radiation when the photo-detection system detectschanged properties, and (iv) computing the presence and position ofcells from the stored positions.

[0016] In a fourth embodiment the invention includes a method fordetermining the presence and position of one or more cells which emitcontrolling radiation comprising: affixing the cell in a measurementregion, wherein controlling radiation emitted in the measurement regionsis incident on a two-dimensional (2D) photosensitive waveguide, andwherein one or more properties of radiation guided through the waveguideare responsive to controlling radiation emitted by cells present in themeasurement region, guiding two or more beams of radiation through the2D photosensitive waveguide at a series of positions so that theintersection of the beams scans substantially all of the 2Dphotosensitive waveguide illuminated by the measurement region,detecting the one or more properties of the beams guided through thewaveguide, wherein presence and position of emitting cells is determinedas the proximity of intersection of the beams when changed properties ofthe beams are detected.

[0017] In a fifth embodiment the invention also includes computerreadable media comprising the encoded program instruction for thecontrollers of the invention.

[0018] In all embodiments it is preferable that the cells being detectedare labeled with a fluorophore, and that the embodiment further comprisea source for activation radiation incident on the measurement region forstimulating the fluorophore to fluoresce. Then, this fluorescence is thecontrolling radiation.

BRIEF DESCRIPTION OF DRAWINGS

[0019] The present invention may be understood more fully by referenceto the following detailed description of the preferred embodiment of thepresent invention, illustrative examples of specific embodiments of theinvention and the appended figures in which:

[0020]FIG. 1A illustrates a side view of an embodiment of the presentinvention.

[0021]FIG. 1B illustrates a side view of another embodiment of thepresent invention.

[0022]FIG. 2 illustrates a perspective view of another embodiment of thepresent invention.

[0023]FIG. 3 illustrates a perspective view of another embodiment of thepresent invention.

[0024]FIG. 4A illustrates a top view of another embodiment of thepresent invention.

[0025]FIG. 4B illustrates a side view of the embodiment shown in FIG.4A.

[0026]FIG. 5 illustrates a flow diagram for the invention.

DETAILED DESCRIPTION

[0027] The principles of the invention are described first with respectto FIGS. 1A-B. Following, several specific preferred embodiments ofthese principles are described along with the methods of use of theseembodiments.

[0028] Principles of Cell Detection

[0029]FIG. 1A side view schematically illustrates one aspect of theprinciples of the present invention. Waveguide 100, illustrated in sideview, includes a core 102 and a photosensitive cladding 104. Thecladding and core materials are selected, as is well known in the art,to have relative indices of refraction at the wavelength of guidedradiation 140 (illustrated here as propagating from left to right) andin view of the thickness of the core so that waveguide 100 functionscorrectly as a waveguide. In the following, waveguide function isprimarily illustrated in the approximation in which light rays arecontained in the core by total internal reflection at the core-claddingboundary. Also well known in the art are more accurate descriptions ofwaveguide function, such as by the equations of electromagnetism. Theart of waveguides is widely described in numerous textbooks andpublication; see, for example, Dorf ed., 1997, The ElectricalEngineering Handbook Second Edition, CRC Press, Boca Raton, Fla. , chap42 (page 1069-1095) and the reference cited therein. The term “light” isused herein to encompass not only visible electromagnetic radiation butalso without limitation at least infra-red and ultra-violet radiation.

[0030] It is also important for the present invention that the core andcladding materials by further selected so that waveguide 100 is“photosensitive”. By “photosensitive” is meant herein that the one ormore aspects of the transmission of the light guided by a waveguide isresponsive to controlling light (“light” is as understood above)incident on the waveguide at an angle such that it penetrates into thecladding, or the cladding and the core. Thus the incident angle of thecontrolling light is greater than the angle of total internal reflectionat the interfaces to the cladding and the core which it must cross;preferably the incident angle is substantially orthogonal to thepropagation direction of the guided light. For example, controllinglight 130 penetrates the waveguide substantially orthogonal to guidedlight 140. Also, the controlling light may be otherwise different thatthe guided light, for example it may have a different wavelength. In apreferred embodiment;it is intensity of the guided light that isresponsive to the controlling light; in other words, in this embodimentthe transmissivity (or absorptivity) of the waveguide is responsive tothe controlling light. In other embodiments, other optical properties ofthe guided light, such as its polarization (or propagation mode) may beresponsive to the controlling light.

[0031] Waveguide photosensitivity is preferably achieved by furtherselecting either the cladding material, or the core material, or bothmaterials to have one or more of their optical properties be responsiveto the controlling radiation (i.e., be a photosensitive material). Inthe embodiment of FIG. 1A, it is the cladding material that isphotosensitive. In this embodiment, selection of the core material iswell known to those of ordinary skill in the art of optical waveguidesand may include, without limitation, transparent organic polymers suchas PMMA or polyacrylate, or transparent inorganic glasses. Selection ofthe cladding material will depend in part on the particular propertyresponsive to the controlling light. Preferably, but without limitation,the refractive index of the cladding is responsive to the controllinglight. Suitable cladding materials are referred to as nonlinear optical(NLO) materials and are described generally in, for example, ChemicalReviews, vol. 94, 1994. A review article providing various classes ofNLO materials suitable for this invention is found in Moemer et al.,1994, Polymeric Photorefractive Materials, Chemical Reviews 94:127-156,and in Henk et al. 1998, SPIE Proc. vol. 2025B:292-297, both hereinincorporated by reference for all purposes.

[0032] Operation of this embodiment, in which the index of refraction ofthe cladding is responsive to the controlling light, is readilyexplained using the internal-reflection approximation of waveguidefunction. Turning again to FIG. 1A, a light beam is confined to awaveguide by multiple total internal reflections of the light beam atthe interface of the waveguide core 102 and the waveguide cladding 102,i.e., the material surrounding the core. Generally, cladding 104 has athickness of at least several guided-light wavelengths, and may be afilm on the core, or may be a surrounding gas or liquid, or may be asolid material adjacent to the core. Since the internal reflections atthe core-cladding interface depend (in part) on the relative index ofrefraction of the core 102 and cladding 104, then if one or both indiceschange in an appropriate manner (to decrease the difference in theindices), the transmissivity of guided light beam 140 will decrease.Simply put, as the difference in the indices of refraction changes, forexample, due to controlling light 130, some of the guided light will“leak” out of the waveguide and the transmissivity will decrease.(Conversely, if the difference in the indices increase, thetransrnissivity may increase.) Changes in the intensity of the guidedlight can be detected and measured.

[0033] The photosensitive waveguides that produce detectable changes inguided-light intensity are exploited in the present invention bycombining them with measurement regions. In operation, a measurementregion contains particles (generally, localized materials) to bedetected that emit controlling radiation, and is arranged with respectto the waveguide so that the emitted controlling radiation penetratesthe cladding, or the core, or both, and causes a detectable change inthe guided light. The measurement region can be separate and adjacent toa waveguide, as in FIG. 1A, or waveguide may form a part of themeasurement regions, as in FIG. 1B. Since a principal application ofthis invention is to detection of biological cells, in the following theterm “cells” is used for both cells in the biological sense and forlocalized particles in general.

[0034] In more detail, FIG. 1A illustrates measurement region 110separate from but adjacent to photosensitive waveguide 100. Measurementregion 110 comprises at least surface 116, which may be glass, plasticor other material, which is suitable for affixing sample 115 of cells toits surface. Affixing cells may be accomplished as is known in the art,for example, by chemical linking or with a surface treatment, byimmunoabsorption on the surface, by coating with a retaining layer (notshown), by being embedded in transparent paraffin (not shown), or soforth. Sample 115, which preferably comprises biological cells and isaffixed to surface 116 (such as a glass slide) comprises “common” cells117 and “rare” cells 118. “Common” and “rare” are used herein todesignate cells not of interest and cells of interest that are to bedetected. These terms are not intended to limit the relative abundancesof the designated cell types, although it is preferable for the rarecells to have a relative abundance of less the 25%, 10%, 5%, 1%, 0.5%,and less. (The methods of the present invention are more efficient andreliable at lower relative abundances of the cells of interest). Forexample, in a sample of maternal peripheral blood, maternal blood cellswould be considered common cells not of interest while fetal cells inthe sample would be considered rare cells of interest.

[0035] The cell sample is further prepared in many ways known in thebiological and biochemical arts so that the rare cells emit controllinglight, for example, by being labeled with a label that emits fluorescentlight of a wavelength to which waveguide 100 is photosensitive. Forexample, the rare cells of interest may be labeled with an antibody toan extracellular or intracellular antigen not present in the commoncells. This antibody may be directly conjugated to a suitablefluorophore, or the cells may be labeled with more fluorophore by one ormore amplification steps, such as, by using anti-antibodies conjugatedto the fluorophore. A suitable fluorophore is a compound whichfluoresces with high quantum efficiency at a wavelength to which thewaveguide is sensitive, and preferably is activated by light of awavelength to which the waveguide is not sensitive.

[0036] Once the rare cells are suitably labeled and sample 115 placed inmeasurement region 110, it is illuminated with activation light 120,which causes the fluorophores labeling rare cell 118 to emit fluorescentlight 130. Since common cells 117 are not so labeled, then do not emitthe fluorescent light 130. The fluorescent light 130 emitted by thefluorophores is then absorbed by cladding 140, and the resulting changesin the index of refraction causes light leakage 141 in the regionadjacent to rare cell 118 and a detectable decrease in the intensity ofthe guided light. Detection of decreased in guided-light intensity ishereinafter referred to as a detection event (or simple an “event”), andrepresents the detection of a rare labeled cell,

[0037] A measurement region may also include certain optional elements.If the waveguide is also sensitive to the activation light of thefluorophore, a filter 112 may be positioned between the measurementregions and the cladding. The filter 112 may be a monochromatic filteror a narrow band filter that transmits fluorescent light but blocksactivation light. A barrier layer (not shown) may optionally bepositioned between the sample 115 and cladding 140 to preventcontamination of the waveguide 100 from the sample 115 and thereby allowreuse of the waveguide 115. In another embodiment, the waveguide is asingle use disposable unit without a barrier layer.

[0038] A waveguide of this invention may also be photosensitive becauseone or more optical properties of the core are suitably photosensitive.For example, the absorptivity of the core may be sensitive to light of afirst range of wavelengths while remaining transparent to light of asecond range of wavelengths. FIG. 1B illustrates such embodiment of thepresent invention where the core 150 is composed of a photosensitivematerial normally transparent to the guided light 140 of the secondrange of wavelengths, but changes its transparency when it absorbs thefluorescent light 130 of the first range of wavelengths. A fluorophorelabeling the rare cells is then chosen to emit fluorescent light in thefirst range of wavelengths. FIG. 1B also illustrates that the waveguideitself may be part of the measurement region. Here, the cladding 155also serves as the sample support. In this case, a filter is notnecessary, since the core is not sensitive to activation light 120.

[0039] In further embodiments, both the core and the cladding may bephotosensitive. Optical properties other than the index of refraction orthe absorptivity may be photosensitive. Additionally, photosensitivewaveguides may be otherwise constructed, or may alter properties of theguided light other than its intensity.

[0040] Specific Two-dimensional Preferred Embodiments

[0041] FIGS. 1A-B, which depict a one-dimensional (1D) cross-section,illustrate that the presence of absence of one or more rare cells in asample can be determined by detecting changes in guided light beam 140.Optionally, by detecting the degree of change, for example, the degreeof attenuation, the number of rare cells may be approximatelydetermined. But, in many cases, it is desirable to further examine rarecells that may be present, for example, by individual microscopicexamination. To perform such examination, the position of rare cellsmust be determined.

[0042] The present invention determines the position of rare cells in asample affixed to a surface by providing a two-dimensional (2D)photosensitive waveguide and by scanning the waveguide in bothdimensions looking for simultaneous detection of the presence of rarecells. In certain alternatives, such 2D scanning may be achieved byscanning two non-parallel light beams. In other alternatives, 2Dscanning may be achieved by scanning with one light beam in onedirection while the waveguide itself is moved along a non-paralleldirection. Finally, in both these embodiments, the activation light mayuniformly illuminate the entire 2D waveguide while two intersectinglight beams scan the illuminated region to detect an event andtriangulation of the intersecting light beams to determine position.However, instead of scanning the light beams guided by the waveguide, infurther alternative, the activation light itself may be focused onto asmall region, a spot, on the waveguide and scanned over the entirewaveguide using a single light beam tracking the movement of theactivation light to detect an event and the position of the activationlight spot to determine position of the event. In the following specificpreferred embodiments of these alternatives are described.

[0043]FIG. 2 illustrates a perspective view of one embodiment of aphotosensitive 2D waveguide scanned by two non-parallel light beams. Thewaveguide 200 comprises a first set of parallel optical fibers 201 fusedto a second set of parallel optical fibers 202 oriented perpendicular tothe first set of fibers. The fused mesh of fibers is coated withcladding material 205 to form the waveguide 200. The core and claddingmaterials are selected to form a photosensitive waveguide, as previouslydescribed. A first light source 210 is positioned to propagate guidedlight 203 along each fiber of the first set of fibers, and a firstdetector 211 is positioned to measure the transmitted light 204 guidedthrough each of the first set of fibers 201. Both the first light source210 and first detector 211 may be mounted on a single support thatsequentially positions the source and detector to measure light guidedthrough each of the fibers in the first set of fibers 201.Alternatively, the light source may be an array of light sources, suchas LEDs, with each LED positioned in front of a single fiber 206, andthe first detector may be an array of detectors, such as CCDs, orientedat the end of each fiber and facing the light source. Such an approachprovides for more rapid detection and localization because it eliminatesthe mechanical scanning. Similarly, a second light source 212 and seconddetector 213 are disposed to transmit and measure the transmitted lightthrough each of the fibers in the second set of fibers 202. The fibersmay have diameters between 1 and 50 micrometers, preferably between 10and 1 micrometers, and most preferably between 5 and 2 micrometers indiameter. The sample is placed on the waveguide 200 (which forms part ofthe measurement region) and illuminated by an activation light.Alternatively, the measurement region may include a separate samplesupport, and optionally an activation light filter. The activation lightcauses the rare cells to emit fluorescent radiation 250 that changes,for example, the index of refraction of the cladding (or theabsorptivity of the core) at an intersection of a fiber from the firstset of fibers and a fiber from the second set of fibers, therebyreducing the transmission of light through each of the affected fibers.Coordinate of the rare cell is determined by calculating the distance tothe affected first fiber along the first set of fibers and the distanceto the affected second fiber along the second set of fibers.

[0044]FIG. 3 illustrates a perspective view of an embodiment similar tothat of FIG. 2, but having a 2D waveguide different from thepreviously-described optical-fiber mesh. The 2D waveguide of FIG. 3 isplanar, constructed from planar core sheet 301 sandwiched betweenphotosensitive cladding sheets 302. These sheets may have thicknesses ofbetween 1 and 50 micrometers, preferably between 10 and 1 micrometers,and most preferably between 5 and 2 micrometers. Their materials areselected so that planar waveguide 300 is appropriately photosensitive,and may be mounted on a substrate for physical support. A firstphoto-detector comprises a first light source 310 oriented to transmitlight in the y-direction 308 and a first detector 312 oriented tomeasure light transmission in the y-direction 308. Both the first lightsource and the first detector are capable of translation in thex-direction 309. A second photo-detector comprises a second light source315 oriented to transmit light in the x-direction 309 and a seconddetector 316 oriented to measure light transmission in the x-direction309. Both the second light source 315 and second detector 316 arecapable of translation in the y-direction 308. These photo-detectors maybe scanned in other geometrically-complete patterns. The light sourcesare such that the light beams have limited lateral spread, for examplethe source may include collimating optics.

[0045] A preferably programmable controller 320 controls means fortranslating (not shown) the first photo-detector in the x-direction 309,means for translating (not shown) the second photo-detector in they-direction 308, and stores the measured values from the first andsecond detectors detects. The controller further detects the presence ofrare cells in beams from the light sources and performs a geometriccalculation to locate each rare cell in the sample. The means fortranslating the light sources and detectors may be standard controllablelaboratory devices as known in the art.

[0046] Specifically, the controller may first perform a calibrationsweep with no sample on the planar waveguide. The controller performs afirst photo-detector calibration by turning on the first light source,measuring the transmitted light with the first detector and storing thetransmitted light values as the controller translates the first lightsource and first detector in the x-direction. The controller thenperforms a second photo-detector calibration by turning on the secondlight source, measuring the transmitted light with the second detectorand storing the transmitted light values as the controller translatesthe second light source and second detector in the y-direction. A sampleis placed in a measurement region adjacent to or on the planar waveguideand illuminated with an activation light source that causes the labeledrare cells to fluoresce, and to thereby change the index of refractionof the cladding in the vicinity of the fluorescing cell. The controller320 performs a first photo-detector scan by measuring the transmittedlight and comparing the value of the transmitted light to thecalibration value for the position corresponding to the present locationof the first detector 312 along the x-direction 309. If the differencebetween the two values is significant, for example, is greater than apreset threshold value, a rare-cell-detection event is declared by thecontroller and the distance along the x-direction is stored by thecontroller 320. The controller 320 performs a second photo-detector scanby measuring the transmitted light and comparing the value of thetransmitted light to the calibration value for the positioncorresponding to the present location of the second detector 312 alongthe y-direction 308. If the difference between the two values aregreater than a preset threshold value, the distance along they-direction is stored by the controller 320. If the sample contains moremany rare cells, the computed location may be ambiguous. This ambiguityis minimized by lowering the relative abundance of rare cells. It may beovercome by techniques such as partially rotating the 2D waveguide andagain performing the scans, where only rare cell locations found in bothwaveguide orientations are true rare-cell locations.

[0047] A further complication arises when two or more events align on asingle light beam. The controller 320 will not be able to distinguishbetween a single event and a plurality of events unless the reduction intransmission per event is known. The relation between transmission andnumber of events can be roughly estimated during a calibration stepwhere a calibration standard is used to determine the transmissionthrough the waveguide as a function of events. The calibration standardcontains markers-of known size and location that emit fluorescentradiation when illuminated by the activation light. As the twophoto-detectors scan the waveguide, the controller correlates themeasured transmission values to the expected number of events based onthe geometry of the calibration standard.

[0048] The ambiguities described above may be eliminated by illuminatingonly a portion of the waveguide with the activation light. Theilluminated region, hereinafter referred to as the illumination spot isthen scanned over the entire waveguide. Only rare cells illuminated bythe illumination spot will emit fluorescent radiation that affects thephotosensitive cladding and thereby affects the transmission of lightthrough the waveguide. The controller 320 controls the movement of theillumination spot and is capable of determining the position of theillumination spot on the waveguide using techniques known to one ofordinary skill in the electromechanical control art. Referring to FIG.3, an event 330 is shown having a location at (X_(a), Y_(a)). The event330 emits fluorescent radiation that is partially absorbed by thephotosensitive cladding in the vicinity of the event and causes the.cladding to change its refractive properties such that the transmissionof light through the waveguide that passes under the event 330 isaltered. If the sample is uniformly illuminated, two photo-detectors arerequired to locate the event 330 because the event 330 could be from anylocation on the waveguide. However, if the sample is illuminated by afocused illumination beam 335, only rare cells located in theillumination spot will emit the fluorescent radiation that alters thecladding properties and affects the transmission of light through thewaveguide. The controller 320 has means to determine the location of theillumination spot on the waveguide and determines the position of anevent. Therefore, only one photo-detector is required to provide for thedetection of the event 330.

[0049]FIG. 4A (depicting a top view) and FIG. 4B (depicting a side view)together illustrate an embodiment of the present invention in which a 2Dphotosensitive waveguide is moved while being a localized region ofactivation light is scanned in an orthogonal direction. In thisembodiment, planar waveguide 400, comprising planar core sheet 401sandwiched between photosensitive cladding sheets 402, is substantiallycircular and is mounted for rotation on support 440. This planarwaveguide may be constructed as in the embodiment of FIG. 3, inparticular, including a supporting substrate. However, instead ofilluminating the entire sample region with activation light as in thepreviously-described embodiments, only a small portion 410 of the regionis illuminated by the activation light 120. Preferably, this portion isof the sized in conformance with the desired accuracy of rare-cellposition determination. Activation light source 450 (with necessaryoptics) is then translated in the radial direction of the rotating diskwaveguide along path XX′ 420. Light source 430 directs light beam 431(of limited lateral dispersion) through waveguide 400 along a pathcoincident with path XX′ 420. A detector 435 disposed opposite the lightsource 430 and oriented to face the light source measures thetransmitted light 436 through the waveguide 400. Programmable controller460 controls light source 430, activation light source 450, means formoving (not shown) the activation light source 450 along path XX′ 420,means for rotating support 440.

[0050] Controller 460 also calculates positions of rare cells in thesample. It receives, processes, and stores the position and rotationangle of the waveguide 400 and the signal from light detector 435.Rare-cell position is then routinely determined from the angle of thewaveguide and the position of activation light when the light detectorobserves an event.

[0051] Methods of the Invention

[0052] The presence and position of rare cells in an appropriatelyprepared sample placed in a measurement region of the devices of thepresent invention are preferably automatically determined. As describedwith respect to the previous embodiments, a preferably-programmablecontroller provides control signals to light sources, light detectors,and the mechanical means for moving the light sources, detectors, andperhaps also the waveguides. A preferred controller includes amicroprocessor and RAM memory for holding software instructions to causethe microprocessor to carry out the methods of this invention. Apreferred controller also includes interfaces to provide control signalsunder program control and user interfaces for control and reporting ofresults. Software instructions for performing this invention's methodsmay be loaded into the controller from computer readable media ofconvenient types, such as magnetic or optical discs, or may bepermanently stored in a ROM memory.

[0053] A preferred embodiment of the methods of this invention aredescribed with reference to FIG. 5. Although FIG. 5 primarilyillustrates controller operation for the embodiment shown in FIG. 4A, itis largely also applicable to the embodiments of FIGS. 2 and 3 withroutine modification. The controller first performs a calibration scan510 by measuring and storing the transmitted light values as a functionof rotation angle of the disk without a sample while rotating the disk afull 360 degrees. For the device of FIG. 2, the controller performs twocalibration scans for each of the light source-detector combination. Thecontroller then waits 520 until an appropriately prepared and labeledsample is placed in the measurement region, which may be adjacent to oron the planar disk waveguide.

[0054] The controller scanning the sample in step 530 by moving theactivation light source to one end of the path XX′, turning on theactivation light 450 and light source 430, rotating the support 440, andtranslating the activation light 450 along path XX′ 420. If a taggedcell is within the illuminated area, the activation light will cause themarker fluoresce. The fluorescent radiation will be partially absorbedby the photosensitive cladding causing the cladding to change itsrefractive index and thereby change the transmitted light through thewaveguide. The controller compares the measured value of transmittedlight from the detector to the calibrated value for the current rotationangle in 540. If the difference between the two values is significant,for example, by being greater than a preset threshold value, an event isdeclared indicating the detection of a rare cell 550. The controllerstores the current rotation angle of the disk and the location of theactivation light source in 560. The controller checks if the whole diskhas been scanned in 570. If the whole disk has not finished scanning,the controller jumps to 540 and continues to rotate the disk, translatethe activation light source, and compare the measured transmission tothe calibrated value.

[0055] If the whole disk has been scanned, the controller, in step 575,determines the location of events, that is of rare cells, from thestored rotation angles and the corresponding storedactivation-light-source location along the XX′ path. These results arethen displayed or reported to a user, and the controller exits.

[0056] The present invention includes other implementations of thesemethods that will be apparent to one of skill in the art. For example,event position can be determined in step 560.

[0057] The invention described and claimed herein is not to be limitedin scope by the preferred embodiments herein disclosed, since theseembodiments are intended as illustrations of several aspects of theinvention. Any equivalent embodiments are intended to be within thescope of this invention. Indeed, various modifications of the inventionin addition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are also intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. A device for detecting the presence of one ormore particles, wherein the particles emit controlling radiation and areplaced in a measurement region, the device comprising: photosensitivewaveguide, wherein one or more properties of radiation guided throughthe waveguide are responsive to controlling radiation emitted byparticles present in the measurement region, and wherein thephotosensitive waveguide comprises a photosensitive organic polymer, anda photo-detection system responsive to the one or more properties of theradiation guided through the photosensitive waveguide, wherein particlesemitting controlling radiation in the measurement region cause changesin the one or more properties of the radiation guided through thewaveguide which are detectable by the photo-detection system, wherebythe device detects the presence of particles.
 2. The device of claim 1wherein the radiation guided through the waveguide comprises infra-redradiation, visible light, or ultra-violet radiation.
 3. The device ofclaim 1 wherein the photo-detection system further comprises a radiationsource for transmitting radiation guided by the waveguide, and adetector for detecting properties of radiation guided through thewaveguide.
 4. The device of claim 1 wherein the photosensitive waveguidefurther comprises a core, and a cladding surrounding the core, whereinone or more optical properties of the cladding are responsive to thecontrolling radiation transmitted into the cladding, wherein the coreand the cladding have relative indices of refraction to form awaveguide.
 5. The device of claim 4 wherein the cladding has an index ofrefraction responsive to the controlling radiation, and wherein thecontrolling radiation decreases the intensity of the radiation guidedthrough the waveguide by causing light to leak from the waveguidethrough the cladding.
 6. The device of claim 1 wherein thephotosensitive waveguide further comprises a core, wherein one or moreoptical properties of the core are responsive to the controllingradiation transmitted into the core, and a cladding surrounding thecore, wherein the core and the cladding have relative indices ofrefraction to form a waveguide.
 7. The device of claim 6 wherein thecore has an absorptivity responsive to the controlling radiation, andwherein the controlling radiation decreases the intensity of theradiation guided through the waveguide by causing light to be absorbedin the core.
 8. The device of claim 1 wherein the photosensitivewaveguide further comprises an inorganic glass.
 9. The device of claim 1wherein the particles emit controlling radiation in response to incidentactivation radiation, and further comprising a source for activationradiation incident on the measurement region.
 10. The device of claim 1wherein the particles are fluorescent, and wherein the source ofactivation radiation stimulates the fluorescence of the particles. 11.The device of claim 1 wherein the fluorescent particles comprise cellslabeled with a fluorophore.
 12. The device of claim 1 wherein thephotosensitive waveguide has a substantially cylindrical shape.
 13. Thedevice of claim 1 wherein the photosensitive waveguide has asubstantially planar shape.
 14. A system for detecting the presence andposition of one or more cells which emit controlling radiationcomprising: a measurement region in which the cells are affixed, atwo-dimensional (2D) photosensitive waveguide, wherein one or moreproperties of radiation guided through the waveguide are responsive tocontrolling radiation emitted by cells present in the measurementregion, and wherein the 2D photosensitive waveguide comprises aphotosensitive organic polymer and a photo-detection system responsiveto the one or more properties of a first beam of radiation and of asecond beam of radiation, wherein the first and second beam are guidedthrough the photosensitive waveguide in non-parallel directions, whereincells emitting controlling radiation in the measurement region causechanges in the one or more properties of the first or the second beam ofradiation when the beams guided through the waveguide in proximity to anemitting cell, the changed properties being detectable by thephoto-detection system, whereby the system detects the presence andposition of cells.
 15. The system of claim 14 wherein the 2Dphotosensitive waveguide further comprises an inorganic glass.
 16. Thesystem of claim 14 wherein the 2D photosensitive waveguide is a mesh ofintersecting optic fibers.
 17. The system of claim 14 wherein the 2Dphotosensitive waveguide comprises a substantially planar core layer andcladding layers surrounding the core layer.
 18. The system of claim 17wherein the planar 2D photosensitive waveguide is disk shaped.
 19. Thesystem of claim 14 wherein at least one of the cladding layers has anindex of refraction responsive to the controlling radiation, and whereinthe controlling radiation decreases the intensity of the radiationguided through the waveguide by causing light to leak from the waveguidethrough the cladding in proximity to the controlling radiation.
 20. Thesystem of claim 14 wherein the core layer has an absorptivity responsiveto the controlling radiation, and wherein the controlling radiationdecreases the intensity of the radiation guided through the waveguide bycausing light to be absorbed in the core in proximity to the controllingradiation.
 21. The system of claim 14 wherein the measurement regioncomprises a surface on which the cells are affixed.
 22. The system ofclaim 21 wherein the surface comprises a surface of one of the claddinglayers.
 23. The system of claim 14 wherein the cells labeled with afluorophore, and further comprising a source for activation radiationincident on the measurement region, wherein the controlling radiation isfluorescent radiation from the fluorophore stimulated by the activationradiation.
 24. The system of claim 14 further comprising: means formoving the first and the second beam of radiation in non-paralleldirections so that their region of intersection scans substantially allof the 2D photosensitive waveguide that is exposed to the measurementregion, and a controller for providing control signals to thephoto-detection system and to the means for moving.
 25. The system ofclaim 24 wherein the controller further comprises: a memory, and aprocessor coupled to the memory and for causing the generation of thecontrol signals, wherein the memory contains encoded programinstructions for causing the processor to perform the steps of (i)generating control signals to cause the means for moving to move thefirst and the second beam of radiation so that their region ofintersection scans substantially all of the measurement region, (ii)generating control signals to cause the photo-detection system to detectthe changed properties of the beams, (iii) storing the positions of thebeams when the photo-detection system detects changed properties, and(iv) computing the presence and position of cells from the storedpositions.
 26. A computer readable media comprising the encoded programinstruction of claim
 25. 27. A system for detecting the presence andposition of one or more cells, wherein the cells are labeled to emitcontrolling radiation in response to incident activation radiation, thesystem comprising: a two-dimensional (2D) photosensitive waveguide,wherein one or more properties of radiation guided through the waveguideare responsive to controlling radiation emitted by cells present in themeasurement region, and wherein the waveguide is planar andsubstantially disk shaped and comprises a photosensitive organicpolymer, and a measurement region in which the cells are affixed, meansfor rotating the disk-shaped 2D planar photosensitive waveguide togetherwith the measurement region, a photo-detection system responsive to theone or more properties of a beam of radiation, wherein the beam isguided through the photosensitive waveguide along a diameter of thedisk-shaped 2D planar photosensitive waveguide, and means for scanning abeam of activation radiation along the path of the beam guided thoughthe waveguide, wherein the activation radiation causes the labeled cellsto emit controlling radiation, wherein cells emitting controllingradiation in the measurement region in response to incident activationradiation cause changes in the one or more properties of the beam ofradiation when the beam guided through the waveguide in proximity to anemitting cell, the changed properties being detectable by thephoto-detection system, whereby the system detects the presence andposition of cells.
 28. The system of claim 27 wherein the cells arelabeled with a fluorophore, and wherein the activation radiationstimulates the fluorophores to fluoresce.
 29. The system of claim 28further comprising a controller for providing control signals to themeans for rotating and to the means for scanning activation radiation.30. The system of claim 29 wherein the controller further comprises: amemory, and a processor coupled to the memory and for causing thegeneration of the control signals, wherein the memory contains encodedprogram instructions for causing the processor to perform the steps of(i) generating control signals to cause the means for rotating and themeans for scanning so that the region of intersection of the beam guidedthrough the waveguide and the activation beam scans substantially all ofthe measurement region, (ii) generating control signals to cause thephoto-detection system to detect the changed properties of the beamguided through the waveguide, (iii) storing the angular position of thewaveguide and the position of the beam of activation radiation when thephoto-detection system detects changed properties, and (iv) computingthe presence and position of cells from the stored positions.
 31. Thesystem of claim 27 wherein the photosensitive waveguide furthercomprises an inorganic glass.
 32. A computer readable media comprisingthe encoded program instruction of claim
 30. 33. A method fordetermining the presence and position of one or more cells which emitcontrolling radiation comprising: affixing the cell in a measurementregion, wherein controlling radiation emitted in the measurement regionsis incident on a two-dimensional (2D) photosensitive waveguide, andwherein one or more properties of radiation guided through the waveguideare responsive to controlling radiation emitted by cells present in themeasurement region, guiding two or more beams of radiation through the2D photosensitive waveguide at a series of positions so that theintersection of the beams scans substantially all of the 2Dphotosensitive waveguide illuminated by the measurement region,detecting the one or more properties of the beams guided through thewaveguide, wherein presence and position of emitting cells is determinedas the proximity of intersection of the beams when changed properties ofthe beams are detected.
 34. The method of claim 33 wherein thecontrolling radiation is fluorescence emitted by fluorophore, thefluorescent emission being stimulated by activation radiation and thecells being labeled with the fluorophore, and wherein the method furthercomprises a step of illuminating the measurement regions with activationradiation.
 35. A device for detecting the presence of one or moreparticles, wherein the particles emit controlling radiation and areplaced in a measurement region, the device comprising: a photosensitivewaveguide, wherein one or more properties of radiation guided throughthe waveguide are responsive to controlling radiation emitted byparticles present in the measurement region, and wherein thephotosensitive waveguide comprises an inorganic glass, and aphoto-detection system responsive to the one or more properties of theradiation guided through the photosensitive waveguide, wherein particlesemitting controlling radiation in the measurement region cause changesin the one or more properties of the radiation guided through thewaveguide which are detectable by the photo-detection system, wherebythe device detects the presence of particles.
 36. A system for detectingthe presence and position of one or more cells which emit controllingradiation comprising: a measurement region in which the cells areaffixed, a two-dimensional (2D) photosensitive waveguide, wherein one ormore properties of radiation guided through the waveguide are responsiveto controlling radiation emitted by cells present in the measurementregion, and wherein the 2D photosensitive waveguide comprises aninorganic glass, and a photo-detection system responsive to the one ormore properties of a first beam of radiation and of a second beam ofradiation, wherein the first and second beam are guided through thephotosensitive waveguide in non-parallel directions, wherein cellsemitting controlling radiation in the measurement region cause changesin the one or more properties of the first or the second beam ofradiation when the beams guided through the waveguide in proximity to anemitting cell, the changed properties being detectable by thephoto-detection system, whereby the system detects the presence andposition of cells.
 37. A system for detecting the presence and positionof one or more cells, wherein the cells are labeled to emit controllingradiation in response to incident activation radiation, the systemcomprising: a two-dimensional (2D) photosensitive waveguide, wherein oneor more properties of radiation guided through the waveguide areresponsive to controlling radiation emitted by cells present in themeasurement region, and wherein the waveguide is planar andsubstantially disk shaped and comprises an inorganic glass, and ameasurement region in which the cells are affixed, means for rotatingthe disk-shaped 2D planar photosensitive waveguide together with themeasurement region, a photo-detection system responsive to the one ormore properties of a beam of radiation, wherein the beam is guidedthrough the photosensitive waveguide along a diameter of the disk-shaped2D planar photosensitive waveguide, and means for scanning a beam ofactivation radiation along the path of the beam guided though thewaveguide, wherein the activation radiation causes the labeled cells toemit controlling radiation, wherein cells emitting controlling radiationin the measurement region in response to incident activation radiationcause changes in the one or more properties of the beam of radiationwhen the beam guided through the waveguide in proximity to an emittingcell, the changed properties being detectable by the photo-detectionsystem, whereby the system detects the presence and position of cells.